Multiplex newborn screening for Pompe, Fabry, Hunter, Gaucher, and Hurler diseases using a digital microfluidic platform

Abstract

Purpose: New therapies for lysosomal storage diseases (LSDs) have generated interest in screening newborns for these conditions. We present performance validation data on a digital microfluidic platform that performs multiplex enzymatic assays for Pompe, Fabry, Hunter, Gaucher, and Hurler diseases.

Methods: We developed an investigational disposable digital microfluidic cartridge that uses a single dried blood spot (DBS) punch for performing a 5-plex fluorometric enzymatic assay on up to 44 DBS samples. Precision and linearity of the assays were determined by analyzing quality control DBS samples; clinical performance was determined by analyzing 600 presumed normal and known affected samples (12 for Pompe, 7 for Fabry and 10 each for Hunter, Gaucher and Hurler).

Results: Overall coefficient of variation (CV) values between cartridges, days, instruments, and operators ranged from 2 to 21%; linearity correlation coefficients were ≥0.98 for all assays. The multiplex enzymatic assay performed from a single DBS punch was able to discriminate presumed normal from known affected samples for 5 LSDs.

Conclusions: Digital microfluidic technology shows potential for rapid, high-throughput screening for 5 LSDs in a newborn screening laboratory environment. Sample preparation to enzymatic activity on each cartridge is less than 3h.

Keywords: 4-MU; 4-MU-α-Gal; 4-MU-α-IDS; 4-MU-α-IDU; 4-MU-α-gluc; 4-MU-β-Gluc; 4-methyl umbelliferone; 4-methylumbelliferyl α-d-galactopyranoside; 4-methylumbelliferyl α-d-glucopyranoside; 4-methylumbelliferyl α-l-iduronate-2-sulfate; 4-methylumbelliferyl α-l-iduronide; 4-methylumbelliferyl β-d-glucopyranoside; ALL; CDC; CLSI; Centers for Disease Control and Prevention; Clinical and Laboratory Standards Institute; DBS; DMSO; Digital microfluidics; Dried blood spot; GAA; GBA; GLA; GalNac; High throughput; IDS; IDU; LSDs; Lysosomal storage disease; Multiplex enzymatic assay; N-acetyl-d-galactosamine; NBS; Newborn screening; QCBP; QCH; QCL; QCM; RFU; acid α-galactosidase; acid α-glucosidase; acid α-l-iduronate-2-sulfatase; acid α-l-iduronidase; acid β-d-glucosidase; advanced liquid logic, Inc.; d-Sac; d-saccharic acid 1,4 lactone; dimethyl sulfoxide; dried blood spot; lysosomal storage diseases; methyl-β-cyclodextrin; newborn screening; quality control base pool, 0% cord blood+100% leukoreduced adult blood adjusted to 50.5% hematocrit on filter paper; quality control high, cord blood adjusted to 50.5% hematocrit on filter paper; quality control low, 5% cord blood+95% leukoreduced adult blood adjusted to 50.5% hematocrit on filter paper; quality control medium, 50% cord blood+50% leukoreduced adult blood adjusted to 50.5% hematocrit on filter paper; relative fluorescence units; β-MBCD.

Figure 1

Schematic of the digital microfluidic cartridge which conforms to a standard microtiter plate dimensions. Each cartridge has 48 sample wells (reservoirs 1–12, rows A–D), 5 reservoirs for reagents (R1–R5) and 5 reservoirs for stop buffer (S1–S5). Droplets dispensed from these reservoirs are 100 nl. All droplet manipulation is automated through a software program that performs dispensing, transport, mixing, incubation, and disposal of droplets as required. A builtin fluorometer reads the fluorescence at the detection window.

Figure 2

Linear regression analysis for all 5 enzymatic assays (Pompe, Fabry, Hunter, Gaucher, and Hurler). The enzyme concentration ranges from QCL to QCH.

Figure 3

Digital microfluidic analysis of normal and affected samples of all 5 enzymatic assays (Pompe, Fabry, Hunter, Gaucher, and Hurler). The line in the middle represents the median.

Figure 3

Digital microfluidic analysis of normal and affected samples of all 5 enzymatic assays (Pompe, Fabry, Hunter, Gaucher, and Hurler). The line in the middle represents the median.

Figure 3

Digital microfluidic analysis of normal and affected samples of all 5 enzymatic assays (Pompe, Fabry, Hunter, Gaucher, and Hurler). The line in the middle represents the median.

Figure 3

Digital microfluidic analysis of normal and affected samples of all 5 enzymatic assays (Pompe, Fabry, Hunter, Gaucher, and Hurler). The line in the middle represents the median.

Figure 3

Digital microfluidic analysis of normal and affected samples of all 5 enzymatic assays (Pompe, Fabry, Hunter, Gaucher, and Hurler). The line in the middle represents the median.

Figure A.1

Sample layout of QC samples in the precision and linearity experiments. For the precision experiments, QCL, QCM, QCH, and QCBP represent different concentrations of cord blood, CAL is calibrant, and EXT is extraction buffer. For the linearity experiments, the numbers in each well represent different concentrations of QCH and QCL samples as detailed in Figure 2.

Figure A.2

Workflow of the digital microfluidic fluorometric enzyme assays for 5 LSDs. The workflow provides a brief overview of the sample preparation, sample loading onto the disposable cartridge, automated analysis, and data analysis using digital microfluidics for LSD screening.